Platelet Derived Growth Factor (PDGF) is a hormonal peptide normally found in human blood platelets and is indispensible for the growth of connective tissue cells, fibroblasts, in synthetic tissue culture media. Although initially isolated from blood serum, subsequent studies have shown this polypeptide is stored in the alpha-grannules of blood platelets, is transported throughout the vascular system by these cells, and is discharged into the serum during blood clotting or after traumatic vascular injury. The concentration of PDGF in normal human blood is approximately 50 nanograms of PDGF per milliliter of sera.
Two forms of PDGF have been isolated, PDGF-I which has a molecular weight of approximately 35,000 daltons and PDGF-II which has a molecular weight of about 32,000 daltons. Both forms of PDGF are biologically active cationic polypeptides, remain stable at 100.degree. C., and have an isoelectric point (pI) of 9.8. Each PDGF form may be reduced with mercaptoethanol into two component polypeptide chains of approximately 13,000 to 14,000 daltons and 17,000 to 18,000 daltons respectively. The component polypeptide chains of each PDGF form are stable in strong acid (pH 2.0), 8N urea, and 4N guanidine-HCl. The reduction of either type of PDGF into its component chains destroys all specific biological activity. None of these reduced component polypeptide chains individually are biologically active as growth promoting factors.
The biological activity of PDGF and protein fractions containing PDGF is measured by the ability of the polypeptide fraction to specifically induce DNA synthesis in confluent Ba1B/C-3T3 (clone A 31) cells. Biologically active PDGF stimulates the synthesis of DNA and increases the rate at which fibroblasts undergo cell division. Assays measuring stimulation activity are performed in the presence of 5% platelet-poor plasma which is necessary for the expression of an optimal response. Plasma which is deficient in platelets does not stimulate the growth of fibroblasts in artificial culture media. Such platelet deficient plasma has been shown to contain little or no PDGF. The addition of PDGF or protein fractions containing some PDGF to such platelet deficient serum, however, restores the ability of such plasma to stimulate DNA replication and cell division of normal fibroblasts in culture. The increase of DNA synthesis is empirically determined either by autoradioagraphy as described in the publications of Scher, Stathakos, Antoniades, Nature (London), 247:279 (1974) and Pledger, Stiles, Antoniades, and Scher, Proc. Natl. Acad. Sci. USA, 74:4481-4485 (1977) or by measuring the uptake of acid-insoluble H.sup.3 -thymidine by the cells in culture as described in the publication of Antoniades, Stathakos and Scher, Proc. Natl. Acad. Sci. USA, 72:2635-2639 (1975). This specific biological activity of PDGF is measured in units defined by convention as the quantity, in picograms, required to induce 50% of the cells in culture to synthesize DNA.
More detailed information regarding the properties and characteristics of PDGF may be found in these publications: Antoniades and Scher, "Growth Factors Derived from Human Serum, Platelets, and Pituitary: Properties and Immunologic Cross-Reactivity", National Cancer Monograph No. 48, Third Decennial Review Conference: Cell, Tissue and Organ Culture, Lake Placid, N.Y., Sept. 13-17, 1976; Antoniades, Stathakos, and Scher, "Isolation of a Cationic Polypeptide from Human Serum that Stimulates Proliferation of 3T3 Cells", Biochemistry, 72:2635-2639 (1975); Scher, Shepard, Antoniades and Stiles, "Platelet-Derived Growth Factor and the Regulation of the Mammalian Fibroblast Cell Cycle", Biochem. Biophys. Acta 560:217-241 (1979); and Kaplan, Chao, Stiles, Antoniades, and Scher, "Platelet Alpha Grannules Containing a Growth Factor for Fibroblasts", Blood 53:1043-1052 (1979).
Two methods for the extraction of PDGF from platelets are presently known and described in the publications of Heldin, Westermark, and Wasteson, "Platelet-Derived Growth Factor: Purification and Partial Characterization", Proc. Natl. Acad. Sci. USA, 76:3722-3726 (1979) and Antoniades, Scher, and Stiles, "Purification of Human Platelet-Derived Growth Factor", Proc. Natl. Acad. Sci. USA, Vol. 76:1809-1813 (1979). The Heldin procedure prepares a platelet lysate and utilizes charge fractionation, hydrophobic chromatography, and two different types of size separation to yield a PDGF containing protein fraction which is approximately 90% pure. This semi-pure protein fraction shows an increase in specific biologically activity of about 8,000 times the specific activity of normal plasma and represents approximately 5% of the original starting material. This method, however, does not allow for unaided visual identification of the polypeptide. The PDGF fraction is identifiable only by using a fluorescent microscope or similar instrument able to detect fluorescent labeled polypeptides. The Antoniades procedure prepares a boiled platelet lysate precipitant and utilizes two different types of charge fractionation and two different forms of size separation to yield a purified homogeneous PDGF having 20,000,000 times the specific biological activity of unfractionated human sera. This homogeneous PDGF represents a total recovery of about 1.5% of the starting quantity of platelet materials. This method, however, requires a loss of some of the PDGF to staining procedures in order to identify which polypeptides constitute the PDGF fraction. It will be appreciated that neither the Heldin nor Antoniades method provides a non-instrument method for the localization of the PDGF fraction which can then be directly recovered as a whole without loss of material for identification purposes.